The present invention relates to specific binding assays and reagents for use therein for the determination of an analyte in a liquid test medium. In particular, the present invention relates to heterogeneous immunoassays for determining haptens employing immobilized hapten reagents for the separation of bound and free forms of a labeled reagent.
Various heterogeneous specific binding assays have been developed which generally involve specific binding interactions between the analyte to be detected, a specific binding partner for the analyte, and a labeled reagent, which can be the same or different as the binding partner for the analyte. When performing such assays, the labeled reagent becomes bound to its corresponding binding partner to generate a bound species, any of the labeled reagent not so bound being the free species, wherein the extent of binding is a function of the amount of analyte present. Where the detectable response of the labeled reagent is essentially indistinguishable in the bound species and the free species, it is necessary to physically separate such bound and free species from each other in order to effectively determine the amount of analyte present. Accordingly, once the bound and free species of the labeled reagent have been separated from each other, the amount of label present in either fraction thereof is determined by measuring the activity of the particular label of the labeled reagent and correlating such activity with the amount of analyte present.
The physical separation of the bound species from the free species can be accomplished in many ways. In the heterogeneous specific binding assay known as the immunometric assay, the labeled reagent comprises a labeled form of an anti-analyte antibody. According to such format, separation of the free labeled reagent from the bound labeled reagent is accomplished by addition of an immobilized form of the analyte under determination or an analog thereof which will bind with the antibody of the free labeled reagent. For example, U.S. Pat. No. 4,200,436 discloses an immunoassay for the detection of antigen employing an immobilized form of the antigen to be measured to separate the bound and free forms of a labeled monovalent antibody to the antigen. The immobilized form of the antigen is prepared by chemically binding or physically adsorbing the antigen to solid supports or carrier materials, such as polysaccharides or plastics, according to methods known in the art.
Similarly, U.S. Pat. No. 4,551,426 discloses a heterogeneous immunoassay for the hapten digoxin employing an immobilized form of ouabain (a digoxin analog) to separate the bound and free forms of an anti-digoxin labeled antibody. The immobilized form of ouabain is prepared by coupling ouabain, either directly or through a spacer arm such as a protein, polyamino acid, or synthetic linker, to a support material, such as beaded agarose, beaded dextran, polyacrylamide, or glass, according to methods known in the art.
Such support materials, however, together with the processes employed to couple the desired analyte or analog thereof (ligand) to such support materials, result in relatively unstable reagents, reagents exhibiting substantial release or leakage of the ligand into the surrounding liquid when used in an immunoassay as heretofore described. Such instability is believed to be the result of an instability in the linkage between the ligand and the support as well as nonspecific binding of the ligand to the support material. Such instability of the linkage and nonspecific binding of ligand results in a substantial amount of the ligand being slowly released or leaching into the surrounding medium. The leaching of the ligand into the test medium primarily occurs as a result of the ligand being nonspecifically bound to internal and external portions of the support material. Although inconvenient, ligand nonspecifically bound to the external surface can be removed by washing the support material with an aqueous wash solution prior to use in an assay procedure. However, ligand nonspecifically bound to internal portions cannot be effectively removed and such internalized ligand leaches out from the interior of the support material and into the liquid test medium where it can be substantially indistinguishable from analyte from a test sample and free to bind to the labeled reagent, resulting in an inaccurate measurement of the amount of analyte actually present in the test sample.
The synthesis and use of support materials, particularly crosslinked polymer supports, having chemical structures which are physiochemically compatible with the backbone structure of a peptide have also been described for use in solid-phase peptide synthesis. In particular, techniques for coupling peptides to a polymer (Stahl, et al., J. Amer. Chem. Soc., Vol. 101(18) p. 5383(1979)]and the crosslinking of various polymers [Varadarajan, et al., Biopolymers, Vol. 22, p. 839 (1983)]using reverse-phase suspension polymerization in aqueous organic solvent mixtures have been employed to obtain favorable swelling properties of such support materials in order to provide increased external and internal reaction sites.
Accordingly, it is an object of the present invention to provide an immobilized hapten reagent which is stable in aqueous solution and which does not slowly release or leach hapten into a surrounding aqueous solution.
Further, it is an object of the present invention to provide a process of covalently binding a hapten moiety substantially only to the surface of a carrier material with substantially no internalization of nonspecifically bound hapten which would otherwise be slowly released or leached into the surrounding medium.
Another object of the present invention is to provide a stable, immobilized hapten reagent for use in an immunoassay for the effective immobilization and separation of the free species of a labeled reagent from its bound species.
It is still a further object of the present invention to provide a highly sensitive liquid immunoassay having a low, analytically insignificant initial background signal for the accurate determination of a hapten or binding analog thereof in a liquid test sample.